Method for improved multiple species blood analysis

Abstract

A lytic reagent composition highly selective in its interactions with the cell membranes of blood cells and also provides a method of using the reagent composition in a semi-automated or an automated system to effect a significantly improved white blood cell differential determination. The lytic reagent composition is characterized by an ability to selectively shrink the white blood cells into the increasing size order of lymphocytes, a midregion consisting mainly of monocytes, other mononuclear cells, some basophils and eosinophils, and neutrophils and can readily be optimized to effect a significantly improved three component separation on the histogram of commercially available semi-automated and automated blood analyzers when compared to the reagents conventionally employed on such instruments. Also provided is a novel blood diluent that exhibits an improved ability to stabilize blood cells without "fixing" the permeability of their membranes, which is critical in the semi-automated analysis or blood cells. This mechanism for membrane stability is particularly effective for maintaining the morphology and size distribution of blood cells for multiple species of animals.

Having described the invention, what is claimed is: 
     
       1. A method for differentiating white blood cells in an automated or semi-automated particle analyzer into at least three distinct subpopulations comprising the steps of: (A) supplying a whole blood sample, a volume of a blood diluent and a volume of a lytic reagent composition to an automated or semi-automated particle analyzing system;   (B) admixing the whole blood sample, the blood diluent and the lytic reagent composition in a manner sufficient to enable the differentiation of at least three subpopulations of white blood cells; and   (C) analyzing the resulting whole blood sample in the automated or semi-automated particle analyzing system to effect at least a three component differential separation of white blood cells; wherein the lytic reagent composition comprises:     an aqueous solution of active lytic reagents consisting essentially of a heterocyclic quaternary ammonium compound which is represented by the general formula ##STR3##  wherein R 1  is a long chain alkyl radical comprising from 8 to 20 carbon atoms and X is an ion selected from the group consisting of halide, sulfate, phosphate and nitrate ions;   wherein the heterocyclic quaternary ammonium compound of said lytic reagent composition is present in a quantity sufficient to effect at least a three component differential separation of white blood cells.   
     
     
       2. A method according to claim 1, wherein the quantity of active lytic reagents in the volume of lytic reagent composition to the volume of whole blood sample ranges from about 0.15 mg active lytic reagents/μl whole blood to about 3.2 mg/μl whole blood. 
     
     
       3. A method according to claim 2, wherein the blood diluent is an isotonic diluent having a predetermined pH and osmolality and comprises at least one organic buffer, a cell membrane stabilizing reagent and an antimicrobial agent comprising a monoaldehyde. 
     
     
       4. A method according to claim 3, wherein the buffer comprises at least one member selected from the group consisting of ADA, MOPS, PIPES, HEPES, BES BIS-TRIS, TES, TAPSO, MES, DIPSO, MOPSO, TRIS and ACES . 
     
     
       5. A method according to claim 3, wherein the monoaldehyde is formaldehyde. 
     
     
       6. A method according to claim 3, wherein said active lytic reagents include at least one 1-dodecyl pyridinium halide. 
     
     
       7. A method according to claim 1, wherein the lytic reagent composition further comprises a chromagen forming reagent. 
     
     
       8. A method according to claim 7, wherein the chromagen forming reagent is potassium cyanide.